The goal of the Human Genome Project is to sequence the human genome which consists of 3.times.10.sup.9 base pairs. Most automated DNA sequencers employ large slab gel configurations with multicolor fluorescence detection. The scanning confocal systems used in connection with detection of slab gels have also been useful in the detection of electrophoretic separations carried out in capillary arrays. Present state of the art confocal detection systems for capillary array electrophoresis have achieved sequencing rates of 25,000 bases/hr (96 samples, 500 bases/sample, 2 hrs/run). However, it is desirable to perform these separations even faster using optimized electrophoretic devices, at which point, detection of these separations becomes more challenging.
Methods for DNA fragment sizing and sequencing on photolithographically fabricated chips have been described in various published articles, for example: Woolley, A. T., and R. A. Mathies (1995), Ultra high speed DNA sequencing using capillary electrophoresis chips, Anal. Chem. 67:3676-3680; and Woolley, A. T., G. F. Sensabaugh, and R. A. Mathies (1997), High-Speed DNA Genotyping using Microfabricated Capillary Array Electrophoresis Chips, Anal. Chem. 69:2181-2186.
In the devices described in these publications, a glass substrate or chip is photolithographically etched to produce channels in the surface of the substrate. These channels are typically 10-50 micron in depth, 50-200 micron in width and can extend for 5-10 cm on the chip, depending on the layout. Then a second substrate is bonded on top of the etched surface to form enclosed channels or capillaries that are suitable for capillary electrophoresis separations. It has been shown that these microfabricated gel-filled channels can be used to perform high quality DNA separations that are 100-fold faster than slab gels and 10-fold faster than conventional capillary separations. It has also been shown that separations of dsDNA fragments, short tandem repeats, DNA sequencing fragments, proteins, amino acids and other chemical analytes can be performed on these chips. In these studies, single channels have been used and the detection has employed laser excited fluorescence detection of fragments in the single channel using conventional confocal or non-confocal microscope configurations.
Multiple channels have been fabricated on a chip and detected by illuminating and imaging the entire chip with a CCD detector, L. B. Koutny, D. Schmalzing, T. A. Taylor & M. Fuchs, Microchip Electrophoretic Immunoassay for Serum Cortisol, Anal. Chem. 68, 18-22 (1996). However, the sensitivity in the latter study was low. It is hard to see how this design can be used to detect large numbers of multiple channels at high speed.
Recently, chips have been fabricated with up to 12 channels (Wooley et al. (1997)). The anode ends of the channels are bundled together for detection and then the bundled channels were detected with a mechanical scan stage that moves the chip past a confocal detector. The linear scanning confocal detection system obtained fluorescence data at a 2 Hz rate. While this method works well to produce high sensitivity detection of multiple channels, it is difficult to see how this approach can be extended to more than 12 channels as the mechanical scanning of the chip past the detector is awkward and the scan rate (limited to 2 Hz) is not fast enough for most fragment sizing and sequencing applications.
The electrophoresis time for DNA sequencing on chips is very short (&lt;10 min.) and consequently the chips must be scanned at rates of at least 10 Hz to provide adequate resolution of the bands. Linear scanners are not suitable for use at high scan rates because of the stresses placed on the motor systems during the stop and reverse phases of the scanning process and because of the time required for reversal.
The advantage of a confocal scanning system is its high numerical aperture, leading to high light collection efficiency, and its ability to limit the light detection to within the fluorescing confocal volume. This dramatically reduces the sensitivity of the system to stray light and also optically sections the capillary chip so as to reject stray light, fluorescence and scatter from the glass or plastic substrate.
In copending application Ser. No. 08/965,738 filed Nov. 7, 1997 incorporated herein by reference, there is described a microfabricated capillary array electrophoresis device or chip. The device includes an array of separation channels formed on a plate with an array of sample reservoirs coupled to the separation channels for introduction of samples into the channels. In one embodiment of the microfabricated capillary array electrophoresis device the channels extend radially or spirally outward from the center of the plate to the sample reservoirs to form a planar array. In another embodiment the planar array comprises a plurality of parallel microfabricated capillaries or channels. There is a need to provide a scanner that can perform high-sensitivity detection of separations on a radial array of capillary separation channels, or on a large array of linearly parallel channels, at a high sampling rate.